PROJECT SUMMARY Mammals, including humans, are generally considered to have lost the capacity to regenerate their limbs after amputation. However, both human and mouse are able to regenerate the extreme tip of their digits through a true regeneration response similar to that observed in amphibians. This regeneration response establishes the foundation on which we can explore methods to stimulate more extensive regeneration in humans. The mouse digit is a good mammalian model because its regeneration process closely resembles that in humans. The extreme distal tip can regenerate via the formation of a bud of undifferentiated cells (a blastema). But amputations and injuries in more proximal level of the digits and limbs usually result in scar wounding. In these clinically more important situations, the inability of blastema formation is the biggest barrier for successful regeneration. In our studies on limb regeneration in the anuran amphibian, Xenopus laevis, and on the normally non-regenerating middle phalanx amputations in mouse, we have demonstrated that transplantation of a fibrin matrix containing limb progenitor cells, in the presence of additional growth factors, can stimulate regeneration of a complete limb in Xenopus and regrowth of bone in mouse digits. However, the overall regeneration of the amputated digit in mouse is still limited. In this project we propose to achieve good regeneration by constructing a vascularizable 3D fibrin scaffold to mimic a regeneration blastema for transplanting to the digit stump after middle-phalanx amputation. We will generate specific progenitor cells from iPS cell lines established in our labs and engineer a 3D fibrin scaffold for transplantation to test our hypothesis that a mimicking regeneration blastema can stimulate regeneration. We will determine the optimal conditions for constructing a regeneration-stimulating matrix and investigate the tissue interactions between the host and donor and within the donor scaffold. The outcome of this project will establish an approach for stimulating digit regeneration, with the engineering of a transplantable, re-vascularizable 3D scaffold containing a combination of multiple types of progenitor cells and growth factors. Since the cell supply is based on iPS cells, a successful outcome will lead directly to the ability to make similar transplants for repair of human digit and limb injuries.